Herpes simplex virus (HSV) is a common human pathogen. HSV can trigger an inflammatory disease in the cornea, known as herpes stromal keratitis (HSK) in which repeated reinfection of the cornea, over time, leads to progressive scarring in the corneal stroma. There are 50,000 new and recurrent cases of HSK each year in the U.S. and HSV is the leading infectious cause of blindness. In epithelial or corneal tissues, HSV enters sensory neurons and travels in neuronal axons from the periphery to sensory ganglia where latency is established. Periodic reactivation of latent HSV produces virus particles that are transported back to the cornea. HSV movement in both directions involves fast axonal transport on microtubules (MT) over very long distances. Motor complexes ferry cellular proteins and membrane vesicles along axonal MT. HSV and other a-herpesviruses have evolved mechanisms to tether virus particles onto these motors so that the viruses move in a rapid, highly directed fashion from the periphery to ganglia, and back again. Our studies will focus on two HSV membrane proteins, gE/gI and US9, that promote directed spread of HSV in the nervous system. Studies in animal models demonstrated that gE/gI functions in both epithelial and neuronal tissues, while US9 functions exclusively in neurons. gE/gI and US9 facilitate outgoing HSV transport (from ganglia to the cornea) but not incoming transport (from the cornea to ganglia). Related to this, we used an HSV US9 mutant to characterize aspects of viral spread in HSK. However, little or nothing is known about the molecular details of how HSV gE/gI and US9 function in axonal transport. Moreover, there is controversy over the form of a-herpesvirus transport in axons, specifically whether viral nucleocapsids or fully assembled enveloped virions are transported toward axon termini. Aim 1 of our proposed studies will examine HSV transport in axons of cultured neurons, attempting to better understand this fundamentally important process. HSV gE/gI and US9 mutants will be used to provide molecular details of how virus transport is organized or staged in nerve cell bodies, the form of virus that is transported, and how gE/gI and US9 promote these processes. Aim 2 will address steps that occur after HSV reaches the extremities of axons. At this stage, virus particles must move into the extracellular space and across junctions formed between neurons and adjacent epithelial cells. This process appears similar to HSV spread across synapses in connected neuronal circuitry. We will investigate these extracellular events as HSV spreads between cultured neurons and epithelial cells or between connected neurons in vitro, and examine the involvement of viral membrane proteins such as gE/gI and US9, as well as gB, gD, gH/gL. Aim 3 will investigate more basic aspects of HSV egress from cells, specifically how HSV crosses the nuclear envelope. Again, virtually nothing is known about nuclear egress and the role of viral glycoproteins in this process. Improved design of anti-virals and vaccines requires a better understanding of how HSV and other a-herpesviruses spread in a highly directed fashion in both epithelial and neuronal tissues.